High-throughput phenotypic screening for functionally characterizing alt. exons

在NCI/NIH最近的工作中，我们应用了分子工程学， 进一步提高CHyMErA编辑效率的策略。我们已经证明， Cas 12 a C端的6个核定位信号刺激编辑效率 通过促进核积累和Cas 12 a核酸酶的稳定化来表达CHyMErA。我们 目前正在应用改进的CHyMErA筛选平台来鉴定替代外显子， 对癌细胞中细胞适应性和增殖的影响。全面删除 将使用CHyMErA筛选平台进行框架保留替代外显子 在一组7种不同的人癌细胞系（A375，HCT 116，MDA-MB-231，A549，Jurkat， K562，HAP 1）。这些屏幕将能够系统地编目核心和 上下文特异性替代健身外显子，沿着提供的功能性的见解， 转录组的复杂性。目的B我们之前已经开发了一种功能强大的 基因组基因座的组合扰动的通用工具，其基于 表达杂合Cas9和Cas 12 a gRNA。此工具已成功申请 脱落和富集细胞适应性筛选。与传统的单电池相比， 在转录组学中，与scRNA-Seq偶联的CRISPR筛选需要捕获非多聚腺苷酸化的DNA。 除了捕获mRNA之外，gRNA还可以将扰动分配给相应的 转录组输出。目前形式的CHyMErA不允许捕获指南 序列使用任何scRNA-Seq平台。因此，我们的目标是将CHyMErA转化为 微扰工具，其适于捕获mRNA转录组和gRNA， 单细胞水平。为了实现这一点，我们将在中间引入捕获序列， Cas9的tracrRNA和Cas 12 gRNA的末端。这些捕获序列将被 在生成scRNA-Seq文库的第一步期间，使用定制的RT条形码化引物。在 平行的、条形码化的聚T RT引物将用于捕获聚腺苷酸化的mRNA。捕获 序列最近已用于基于Cas9的CRISPR平台，并可用于 使用3 Direct Capture 10 x Genomics平台。我们的目标是将这些捕获序列用于我们的 系统，但需要确保其纳入不影响编辑效率， CHyMErA.为了测试scCHyMErA对于组合CRISPR扰动的适用性， 到scRNA-Seq，我们将使用双靶向hgRNA产生靶向450个基因的小文库， 引导Cas9和Cas 12 a靶向相同的基因。两个独立的hgRNA将被 每个基因设计1,000个文库元件（包括对照）， 按照我们在本书中掌握的方案， 过去对于我们的第一次筛选，我们将重点放在靶向基因编码的RNA结合蛋白 （RBP），其调节RNA加工事件，包括选择性剪接和 聚腺苷酸化这些过程是重要的细胞命运决定及其中断的基础 与包括恶性血液病在内的多种疾病有关。hgRNA文库将在 转导到胚胎干细胞中，随后进行单细胞RNA-Seq 使用标准的10 x基因组学方案进行分析。 在NovaSeq 6000 S2流动池上产生40亿个读数，等于每个分析的50 k读数 细胞和数据将使用已建立的计算管道进行分析。这些数据将不会 不仅将scCHyMErA-Seq建立为scRNA-Seq读出的CRISPR干扰工具，而且 可能揭示在干细胞中起重要作用的基因和RNA相关通路， 维持和多能性。未来的筛选将集中在靶向组织调节 用scCHyMErA-Seq替换盒式外显子，以揭示转录后的 发展轨迹背后的拼接事件。

Aim A In recent work performed at NCI/NIH, we have applied molecular engineering strategies to further boost the editing efficiency of CHyMErA. We have shown that addition of 6 nuclear localization signals at the C-terminus of Cas12a stimulates the editing efficiency of CHyMErA by promoting nuclear accumulation and stabilization of Cas12a nuclease. We are currently applying the improved CHyMErA screening platform to identify alternative exons that impact on cell fitness and proliferation in cancer cells. Comprehensive deletion of frame-preserving alternative exons will be performed using the CHyMErA screening platform across a panel of 7 diverse human cancer cell lines (A375, HCT116, MDA-MB-231, A549, Jurkat, K562, HAP1). These screens will enable the systematic cataloguing of the core and context-specific alternative fitness exons, along with providing insights into the functional complexity of the transcriptome. Aim B We have previously developed CHyMErA, a powerful and versatile tool for combinatorial perturbation of genomic loci, which is based on the expression of hybrid Cas9 and Cas12a gRNAs. This tool has been successfully applied for dropout and enrichment cell fitness screens. Compared to conventional single-cell transcriptomics, CRISPR screens coupled to scRNA-Seq require the capture of non-polyadenylated gRNAs in addition to the capture of mRNA in order to assign perturbations to consequential transcriptomic outputs. CHyMErA, in its current form, does not allow capturing of guide sequences using any scRNA-Seq platform. Therefore, we aim to transform CHyMErA to a perturbation tool that is amenable to capturing both the mRNA transcriptome and gRNAs at the single-cell level. To achieve this, we will introduce capture sequences in the middle of the tracrRNA of Cas9 and at the end of Cas12 gRNAs. These capture sequences will be recognized by customized RT barcoded primers during the first step of generating scRNA-Seq libraries. In parallel, barcoded polyT RT primers will be used to capture polyadenylated mRNA. Capture sequences have recently been used for a Cas9-based CRISPR platform and made available for use with the 3 Direct Capture 10x Genomics platform. We aim to use these capture sequences in our system but need to ensure that their incorporation does not impact the editing efficiency of CHyMErA. To test the suitability of scCHyMErA for combinatorial CRISPR perturbations coupled to scRNA-Seq, we will generate a small library targeting 450 genes using dual-targeting hgRNAs directing both Cas9 and the Cas12a to target the same gene. Two independent hgRNAs will be designed per gene resulting in 1,000 library elements (including controls) that will be cloned as a pool into a lentiviral backbone following protocols that we have mastered in the past. For our first screen we will focus on targeting genes encoding RNA binding proteins (RBPs) which regulate RNA processing events including alternative splicing and polyadenylation. These processes underlie important cell fate decisions and their disruption is linked to numerous diseases including hematological malignancies. The hgRNA library will be transduced into embryonic stem cells and subsequently subjected to single-cell RNA-Seq analysis profiling using standard 10x Genomics protocols.The transcriptomes will be sequenced on a NovaSeq 6000 S2 flow cell resulting in 4 billion reads equal to 50k reads per profiled cell and data will be analyzed using established computational pipelines. These data will not only establish scCHyMErA-Seq as a tool for CRISPR perturbation with scRNA-Seq readout but also may uncover genes and RNA-related pathways that play an important role in stem cell maintenance and pluripotency. Future screens will be focused on targeting tissue-regulated alternative cassette exons with scCHyMErA-Seq in order to uncover the postranscriptional splicing events that underlie developmental trajectories.